The cytokine, interleukin 12 (IL-12), discovered by Giorgio Trinchieri in 1989 [1], bridges the innate and adaptive immune responses by inducing interferon-γ (IFN-γ) production primarily from natural killer and T cells. Cancer therapy with IL-12 exploits its natural immune functions to polarize T cells to the Th1 phenotype, boost effector T cells, downregulate angiogenesis, remodel the extracellular matrix, and alter the levels of immune suppressive cytokines [2]. Due to these activities, IL-12 is one of the most promising cytokines for immunomodulatory cancer therapy.
The initial clinical trials with IL-12 resulted in grave toxicities including deaths, which severely downgraded the reputation and potential application of this effective cytokine. In reality, most anticancer drugs or biological modalities are associated with systemic toxicity. It is desirable to decrease this toxicity to effectively and safely treat the extremely high numbers of cancer patients [2].
A popular strategy for sequestering the effects of cytokine therapies in the tumor environment is targeting cellular markers that are upregulated exclusively in the tumor cells or the tumor microenvironment. Indeed, conjugating IL-12 to tumor-specific antibodies, such as L19 [3] and HER2 [4], and tumor vasculature-specific peptides, such as RGD [5] and CNGRC (SEQ ID NO.9) [6], improves the efficacy of treatments; however, the necessarily high frequency of administrations of recombinant cytokines increases the immunogenicity, toxicity, and cost of treatments. A gene therapy approach would reduce these limitations.
Intratumoral IL-12 gene therapy is able to eradicate 40% of tumors in a murine squamous cell carcinoma model (SCCVII) while systemic delivery via intramuscular administration fails to eradicate any tumors [7]; however, direct injection into tumor sites is rarely available noninvasively or post-surgically. Several methods have been developed to target the IL-12 effect to the tumor after systemic delivery. For example, modifying viral vectors with tissue specific gene promoters such as the CALC-I promoter [8], capsid-expressed tumor-specific peptides [9], and polyethylene glycol or other nanoparticles [10, 11] increases tumor specific expression and decreases systemic expression; however, the fenestrated vasculature of the tumor environment allows for the gene products to leak out of the tumor environment leading to systemic toxicities [12]. Therefore, a gene product that can interact with and remain in the tumor environment will increase the level of therapeutic efficacy and decrease systemic toxicity.
Tumor targeting can be achieved via the screening of various libraries to select tumor-targeted peptides, DNA/RNA aptamers, antibodies, etc; however, the only mechanism that can be used for homing gene products from systemically injected genes will be tumor-targeted mini-peptides encoding DNA. The small size of these peptides eliminates the concern of immunogenicity, and reduces the effect on the biological function of the gene product, though some minipeptides may boost or inhibit gene function [20]. The tiny peptide encoding DNA sequences can be easily fused with any therapeutic gene. Finally, these peptides can complement existing tumor targeting approaches such as transcriptional targeting [8], translational targeting [21], and targeted delivery [3-6].
Currently, most tumor-targeting strategies are based on extremely specific interactions, and the ability to target the tumor environment is constrained to a single cell type or specific type of tumor. Proteins are conjugated with polyunsaturated fatty acids, monoclonal antibodies, folic acid, peptides, and several other chemicals to increase the tumor-targeted ability of the therapeutic protein. Other tumor targeting peptides can deliver small molecules with only one copy for each small-molecule payload but require multiple copies of the peptide to target larger molecules such as a full length cytokine [24].